A highly significant percentage of the genomes sequenced thus far are thought to encode polytopic transmembrane proteins which catalyze a multitude of essential cellular functions, energy and signal transduction in particular. Many are important with regard to human disease (e.g. cystic fibrosis, drug resistance), and many widely prescribed drugs (eg. Prozac and Prilosec) are targeted to membrane transport proteins. Although progress over the last 20 years has led to the characterization, purification and modification of this class of proteins, only a few have been studied at a level useful for understanding mechanism. Furthermore, many membrane proteins require conformational flexibility in order to function, making it imperative to obtain dynamic structural information. The objectives of this application are to continue to utilize the lactose permease of Escherichia coli as a paradigm for structure/function studies on transmembrane proteins. Only 6 amino acid residues are irreplaceable with respect to mechanism, and application of novel site-directed biochemical and biophysical approaches has yielded a helix packing model to a resolution approximating 4 Angstrom units. Further efforts will be made to refine and extend the structure using these methods. In addition, newly developed approaches using site-directed fluorescence resonance energy transfer and solid-state 19F-NMR will be introduced. Ligand-induced conformational changes in certain helices can also be demonstrated, and these studies will be extended to the remainder of the molecule in order to delineate overall structural changes that result from ligand binding. The substrate binding site is located at the interface between helices IV and V, and specificity is directed towards the galactosyl moiety of the substrate. A spin-labeled galactoside that binds to the permease with high affinity has been synthesized and will be used to further define the substrate binding site. Ligands that bind but are not translocated are also being synthesized in order to study binding from the inner and outer surface of the membrane in the absence of translocation. Site-specific alkylation combined with mass spectrometry will be used to determine changes in the protonation of His322 (helix X) upon ligand binding.